Pneumocystic carinii (Pc) organisms cause a lethal pneumonia (PcP) in hosts with compromised immune systems, especially in patients with AIDS. Despite prophylactic, therapeutic, and supportive strategies, PcP remains a major concern in this patient population. To date, no drug has been proven more effective than trimethoprim sulfamethoxazole or pentamidine isethionate for the treatment of PcP. More effective use of existing treatment or identification of new drugs is needed to improve the outcome of PcP. The identification of new compounds for the treatment of PcP has been severely hindered by the lack of a reliable in vitro screening system. During the tenure of the previous grant period, the applicant developed an ATP bioluminescent assay to assess the viability of rat Pc populations. The ATP system: 1) could maintain the metabolic activity of Pc throughout the period of assay; 2) was a rapid and reproducible method; and 3) preliminary drug screens correlated with in vivo activity. The applicant will use this assay to assess new approaches to Pc treatment, adapt it for rapid drug screening, and explore questions of variation in drug susceptibility and resistance in Pc populations. The aims of this project will be to: 1) apply the ATP bioluminescent assay for the purpose of in vitro drug screening. This system will have potential clinical application as a screening system and tracking assay for drug susceptibilities of Pc isolates; 2) explore pentamidine resistance in rat Pc populations; and 3) apply the in vitro system to probe the energy metabolism of Pc and evaluate agents targeted at these pathways.